Development of a nuclear transformation system for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and genetic complementation of a mutant strain, deficient in arachidonic acid biosynthesis.

Boris Zorin,Sammy Boussiba,Olivier Vallon,Nicolas Tourasse,Omer Grundman,Michal Shapira,Yuval Kaye,Inna Khozin-Goldberg,Stefan Leu,Marie-Joelle Virolle

doi:10.1371/journal.pone.0105223

Abstract

Microalgae are considered a promising source for various high value products, such as carotenoids, ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The unicellular green alga Lobosphaera (Parietochloris) incisa is an outstanding candidate for the efficient phototrophic production of arachidonic acid (AA), an essential ω-6 PUFA for infant brain development and a widely used ingredient in the baby formula industry. Although phototrophic production of such algal products has not yet been established, estimated costs are considered to be 2–5 times higher than competing heterotrophic production costs. This alga accumulates unprecedented amounts of AA within triacylglycerols and the molecular pathway of AA biosynthesis in L. incisa has been previously elucidated. Thus, progress in transformation and metabolic engineering of this high value alga could be exploited for increasing the efficient production of AA at competitive prices. We describe here the first successful transformation of L. incisa using the ble gene as a selection marker, under the control of the endogenous RBCS promoter. Furthermore, we have succeeded in the functional complementation of the L. incisa mutant strain P127, containing a mutated, inactive version of the delta-5 (Δ5) fatty acid desaturase gene. A copy of the functional Δ5 desaturase gene, linked to the ble selection marker, was transformed into the P127 mutant. The resulting transformants selected for zeocine resistant, had AA biosynthesis partially restored, indicating the functional complementation of the mutant strain with the wild-type gene. The results of this study present a platform for the successful genetic engineering of L. incisa and its long-chain PUFA metabolism.

Highlights

The green freshwater microalga Lobosphaera incisa (Reisigl) comb. nov. was isolated from snow water patches in the alpine environment of Mt
arachidonic acid (AA) constitutes about 60% of total fatty acids (FA) (TFA), and over 90% of cell AA is deposited in TAGs, making L. incisa the richest plant source of the pharmaceutically and nutraceutically valuable AA and a target organism of high biotechnological interest [3] [4]
In order to improve the biotechnological potential of L. incisa, we developed a stable nuclear transformation system for its metabolic engineering

Summary

Introduction

The green freshwater microalga Lobosphaera incisa (Reisigl) comb. nov. was isolated from snow water patches in the alpine environment of Mt. Was isolated from snow water patches in the alpine environment of Mt. Tateyama, Japan [1]. It belongs to the Trebouxiophyceae, a class of Chlorophyte algae. Lobosphaera incisa was initially assigned to the genus Parietochloris rather than to Myrmecia [1] but was recently reclassified as belonging to Lobosphaera based on zoospore morphology and 18S rDNA analysis [2]. When cultivated under nitrogen starvation, the total FA content of the alga is over 35% of dry weight. AA constitutes about 60% of total FAs (TFA), and over 90% of cell AA is deposited in TAGs, making L. incisa the richest plant source of the pharmaceutically and nutraceutically valuable AA and a target organism of high biotechnological interest [3] [4]

Methods

Results

Conclusion